摘要
目的观察过氧化物酶体增殖物活化受体1配体吡格列酮对高糖作用下大鼠腹膜间皮细胞（RPMC）合成细胞外基质的作用以及调节机制。方法胰蛋白酶消化法分离培养RPMC。随机分为正常对照组、高糖组（2．5％葡萄糖）、吡格列酮干预组（10μmol／L、20μmol／L＋2．5％葡萄糖）、二硫氨基甲酸吡咯烷干预组（PDTC，NF—KB抑制剂，25tzmol／L、50μmol／L＋2．5％葡萄糖）、姜黄素干预组『活化蛋白1（AP-1）抑制剂，15μmol／L、30μmol／L＋2．5％葡萄糖1。RT—PCR方法检测纤连蛋白（FN）、I型胶原（COLI）、纤溶酶原激活抑制因子1（PAI-1）、c—fos、c—junmRNA表达。ELISA方法检测细胞上清液中FN、COLI和PAI-1蛋白水平。Western印迹方法检测IKB（x、磷酸化IκBα（p-IκBα）、NF—κBp65、磷酸化NF—κBp65（p-p65）蛋白表达。结果常规培养的腹膜间皮细胞表达基础量的FN、COLI和PAI-1，高糖显著上调其蛋白及mRNA表达（P〈0．01）。吡格列酮预处理后，高糖诱导的FN、COLI和PAI-1蛋白及mRNA表达显著低于高糖组（P〈0．01）。高糖作用后，磷酸化IKBα和NF—κBp65水平显著增高，c—fos、c—junmRNA表达增加，与对照组差异有统计学意义（P〈0．01）。PDTC预处理后，高糖诱导RPMC的FN和PAI-1蛋白水平降低（P〈0．01），COLI蛋白表达无明显变化。AP-1抑制剂姜黄素预处理后，高糖诱导的RPMCFN、COLI和PAI-1蛋白水平均显著降低，与对照组差异有统计学意义（P〈0．01）。吡格列酮抑制高糖条件下磷酸化IKBα和NF-κBp65水平，抑制c—fos、c—junmRNA表达（P〈0．05或P〈0．01）。结论NF—κB和AP-1信号通路参与高糖条件下RPMC的FN、COLI和PAI-1表达的调节。吡格列酮通过NF—κB和AP-1途径下调高糖诱导的RPMC的FN、COLI和PAI-1的表达，从而发挥抗纤维化作用。
Objective To study the effect and underlying mechanism of pioglitazone （PPARγ, ligand） on increased extracellular matrix （ECM） production in rat peritoneal mesothelial cells （RPMCs） induced by high glucose. Methods RPMCs were isolated and subeuhured by enzymatic disaggregation. The cells were randomly divided into groups as follows： normal control group,high glucose group （2.5%glucose）, PDTC group （NF-KB inhibitor, 25 μmol/L and 50 μmol/L＋ 2.5%glucose）, curcumin group （activator protein-1 inhibitor, 15 μmol/L and 30 μmol/L＋2.5% glucose） and pioglitazone group （10 μmol/L and 20 μmol/L＋2.5%glucose）. RT-PCR was used todetect the mRNA expression of fibronectin （FN）, collagen I （COL I ）, plasminogen activator inhibitor-1 （PAI-1）, c-fos and c-jun. The protein expression of p-IKBα, IKBα, p-p65, NF-KBp65 was estimated by Western blotting. Concentrations of FN, COL I and PAI-1 in culture medium were examined by ELISA. Results The expressions of FN, COL I and PAI-1 in RPMCs were significantly increased after treatment with high glucoce （P〈0.01） and significantly decreased after treatment with pioglitazone （P〈0.01）. High glucose up-regulated th phosphoralation of IKBα, NF-κBp65 and the expression of c-fos and c-jun mRNA. Both PDTC and curcumin reduced the protein expression of FN and PAI-1 in RPMCs induced by high glucose （P〈0.01）, not PDTC but curcumin decreased the protein expression of COL I （P〈0.01）. Pioglitazone partially reversed the effect of high glucose on the phosphoralation of IKBα NF-κBp65 and the mRNA expression of c-fos and c-jun （P〈0.01）. Conclusion Pioglitazone can suppress the increased ECM production in RPMCs induced by high glucose, and this effect might be related to the inhibitory effects on activation of NF-κB and activator protein-1 pathways.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2010年第3期198-203,共6页
Chinese Journal of Nephrology
